New blood vessel formation, angiogenesis, is required for normal development and wound healing. Aberrant angiogenesis contributes to many diseases including tumor growth, diabetic retinopathy, arthritis and psoriasis. Endothelial cell migration is one of the critical steps in angiogenesis and is promoted by angiogenic stimulating factors such as VEGF. Our early studies demonstrate that, under normaxic conditions, the activity of MAPK-activated protein kinase 2 (MK2) is required for VEGF- stimulated endothelial cell migration and that MK2 participates in endothelial cell migration by regulating urokinase plasminogen activator (uPA) expression. As angiogenesis occurs in the hypoxic environment, we investigated the involvement of MK2 and uPA in endothelial cell migration under the hypoxia. We show that, similar to what we have observed in normaxia, inhibiting MK2 activity also abrogates uPA expression and VEGF-stimulated endothelial cell migration while restoring uPA expression prevents MK2 inhibitor-caused inhibition in endothelial cell migration under hypoxia. These findings demonstrate a general role of the MK2-uPA axis in endothelial cell migration under both normaxia and hypoxia. In an effort to define the mechanism by which MK2 regulates uPA expression, we found that the activity of MK2 is important for relatively stable uPA mRNA in endothelial cells. Through a two-hybrid screening, we identified an RNA binding protein DDX5 that not only specifically interacts with MK2 but also serves as a direct substrate of MK2. Overexpression of DDX5 destabilizes uPA mRNA and silencing DDX5 expression prolongs the half-life of uPA mRNA in MK2-inhibited cells. DDX5 directly interacts with uPA mRNA and the degree of DDX5-uPA mRNA interaction is negatively regulated by MK2 activity. These results suggest that the MK2 may stabilize uPA mRNA by preventing DDX5 to interact with uPA mRNA and thus impeding DDX5's ability to mediate uPA mRNA decay. In our latest studies, we further investigated the potential role of the exosome in DDX5-mdiated uPA mRNA degradation. DDX5 interacts with the exosome in MK2-inhibited cells and knocking down the expression of the exosome subunits prolongs uPA mRNA stability in MK2-inhibited or DDX5- overexpressed cells. These results firmly link the exosome to MK2-DDX5 regulation of uPA mRNA stability. This proposal is to capitalize on our previous work and contains three aims: 1) determine how MK2 prevents DDX5 from facilitating uPA mRNA turnover;2) determine the mechanism associated with DDX5-exosome interaction and its role of the exosome in uPA mRNA degradation;and 3) determine the effectiveness of intercepting the MK2-DDX5-uPA axis for suppressing angiogenesis. The proposed studies should increase our understanding of how endothelial cell migration is regulated, and may also help to develop a novel therapeutic approach to suppress pathological angiogenesis. PUBLIC HEALTH RELEVANCE New blood vessel formation, or called angiogenesis, is required for normal development and wound healing. Aberrant angiogenesis contributes to many diseases including tumor growth, diabetic retinopathy, arthritis and psoriasis. This application focuses on one of the critical steps of angiogenesis, directional endothelial cell migration. In our published studies and studies presented in this application, we found that a protein called MAPK-activated protein kinase 2 (MK2) is required for directional migration of endothelial cells in patho/physiological condition (hypoxia), and that MK2 participates in endothelial cell migration by regulating urokinase plasminogen activator (uPA) expression. To understand MK2 regulation of uPA expression, our preliminary studies revealed that MK2 promotes the levels of uPA by preventing DDX5 to interact with uPA mRNA and the exosome (consisting of RNA enzymes) and thus prolonging uPA mRNA stability. These findings demonstrate a novel mechanism involving MK2-DDX5 axis to regulate uPA level and endothelial cell migration. In this application, we wish to further investigate the functional link among MK2, DDX5 and uPA mRNA turnover. We also wish to employ the knowledge obtained from these studies to develop a therapeutic approach for inhibiting pathological angiogenesis.